Dissertation > Biological Sciences > Bioengineering ( Biotechnology ) > Enzyme Engineering

Purification by MCAC and Molecular Immobilization of SOD from Maize

Author LiuJianLi
Tutor ChenHongMan
School Shenyang Agricultural University
Course Biochemistry and Molecular Biology
Keywords Superoxide dismutase Metal chelate affinity chromatography Chemical modification : drug-loaded microspheres Immobilization Encapsulation efficiency
CLC Q814
Type Master's thesis
Year 2005
Downloads 224
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Corn grain as raw material by ammonium sulfate fractionation, gel filtration, and metal chelate affinity chromatography-step purification of corn SOD. After electrophoresis, activity staining and protein staining were obtained three bands, specific activity 8597U/mg, purification factor of 307, the recovery rate of 29.4 percent to electrophoretic homogeneity. Characterization studies have shown that the molecular weight of SOD subunit 16.0KD, maximum UV absorption of 260nm, the enzyme at pH 5-11,80 ℃ stable within the following ranges, corn SOD on KCN and H 2 O 2 sensitive to Vmax of pyrogallol as 49.50mmol/min, Km is 0.1634mmol / L. For SOD as a medicinal enzyme deficiencies, this paper SOD were modified with immobilization. First, a PEG of SOD for the chemical modification. Modification rate of 46%, SOD activity retention was 81%. Modification of SOD after temperature stabilization, pH stability are significantly improved, compared with the natural SOD, PEG-SOD trypsin digestion significantly enhanced tolerance. The results showed that, PEG modification of SOD is feasible, PEG SOD is a better chemical modifier. To enhance cell permeability of SOD, this study adopts rotary film - Ultrasonic SOD prepared saturated lecithin small unilamellar liposomes were determined using the method of SOD activity encapsulation efficiency, determined by orthogonal experimental design optimal Preparation conditions: saturated lecithin and cholesterol ratio of amount of substance (A) is 6:7, the temperature (B) is 50 ℃, ultrasonic treatment time (C) is 4.5min, pH of the enzyme solution (D) is 6.8, Under these conditions, the encapsulation efficiency was 48.14%. Compared with the natural SOD, SOD in liposomes little change in physical and chemical properties, in pH5-11, 80 ℃ less stable in the range. TEM results showed that: particle size distribution 77 ± 21nm. Purified in our laboratory corn SOD, racemic d, 1 - polylactic acid as the carrier, using the double emulsion solvent evaporation prepared SOD-polylactic acid microspheres investigated by orthogonal experimental design polylactic acid concentration, SOD enzyme amount , polyethylene glycol concentration, the amount of emulsifier, and the encapsulation efficiency of particle size, to determine the optimal conditions for preparing microspheres and investigate physical and chemical properties of SOD microspheres. The results show that the double emulsion solvent evaporation method has been high encapsulation efficiency, particle size and distribution than satisfactory SOD polylactic acid microspheres. Compared with the natural SOD, SOD microspheres is essentially the same optimum temperature and pH increased temperature

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